1. Field of the Invention
The present invention provides for fusion proteins capable of synchronously binding vascular endothelial growth factor (VEGF) and angiopoietin, namely “double anti-angiogenic proteins (DAAP)”. DAAP are disclosed which are therapeutically useful for treating VEGF and angiopoietin-associated conditions and diseases such as cancer, age-related macular degeneration, diabetic retinopathy, rheumatoid arthritis, psoriasis, acute and chronic inflammations, arteriosclerosis and lymphatic proliferative diseases.
2. Description of the Background
There are three known VEGF receptors, VEGFR1, VEGFR2 and VEGFR3 in humans. Human VEGFR1 consists of 1338 amino acids, separated by three major regions: an extracellular domain consisting of seven immunoglobulin (Ig)-like domains, a transmembrane domain and an intracellular tyrosine kinase domain (UniProtKB/Swiss-Prot entry P17948) (FIG. 1). VEGFR2 and VEGFR3 are similarly organized and display about 80% identity to VEGFR1 in the tyrosine kinase domain.
Activation of VEGFR1 and VEGFR2 by binding of VEGF-A plays a crucial role for growth, migration and survival of blood endothelial cells, which are essential processes for angiogenesis and vasculogenesis, whereas activation of VEGFR3 by binding of VEGF-C and VEGF-D plays a main role for growth, migration and survival of lymphatic endothelial cells, which are essential processes for lymphangiogenesis (Shibuya M and Claessson-Welsh L, Exp. Cell Research 312:549-560, 2006; Alitalo K, et al., Nature 438:946-953) otherwise specifically indicated.
Affinity of VEGF-A to VEGFR1 is approximately 10 times higher than that to VEGFR2. Among 71 g-like domains of VEGR1, the Ig-like domain 2 is essential for VEGF-A binding (FIG. 2). However, the Ig-like domain 2 of VEGFR1 contains many basic amino acids and its theoretical isoelectric point (pI) is 9.19 (Compute pI/Mw tool for Swiss-Prot/TrEMBL entries, http://kr.expasy.org/tools/pi_tool.html). Therefore, the Ig-like domain 2 of VEGFR1 per se cannot be used as therapeutic protein with property of decoy receptor because it has low pharmacokinetic properties.
Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) is a receptor tyrosine kinase (RTK) expressed predominantly on endothelial cells and hematopoietic cells (Dumont D J, et al., Oncogene 8: 1293-1301, 1993). Tie2 is critical for vasculogenesis, angiogenesis, and hematopoiesis (Yancopoulos G D, et al., Nature 407:242-248, 2000). Four Tie2 ligands have been identified: angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), angiopoietin-3 (Ang3), and angiopoietin-4 (Ang4) (Yancopoulos G D, et al., Nature 407:242-248, 2000). Although Ang1 seems to be an obligate activator of Tie2, Ang2 seems to have context-specific effects, activating this receptor on some cells while blocking Tie2 activation on other cells or under different conditions (Yancopoulos G D, et al., Nature 407:242-248, 2000).
Human Tie2 consists of 1124 amino acids, separated by three major regions: an extracellular domain consisting of two Ig-like domain, three EGF-like domain, one Ig-like domain, three fibronectin type-III; a transmembrane domain and an intracellular tyrosine kinase domain (UniProtKB/Swiss-Prot entry Q02763) (FIG. 1).
Among extracellular subdomains of Tie2, the Ig-like domain 2 is essential for angiopoietin binding, but the Ig-like domain 1 and three EGF-like domain appear to be required for stable binding of angiopoietin (FIG. 2). Importantly, these subdomains contain many acidic amino acids and its theoretical isoelectric point (pI) is 6.55 (Compute pI/Mw tool for Swiss-Prot/TrEMBL entries, kr.expasy.org/tools/pi_tool.html). Therefore, these domains per se could be used as therapeutic protein because it could have a high pharmacokinetic value.
Of VEGF and angiopoietin family proteins, VEGF-A and angiopoietin-2 (Ang2) are critical molecules for tumor angiogenesis (Holash, J. et al., Science 1999; 284:1994-1998; Holash, J. et al., Oncogene 1999; 18:5356-5362) and metastasis (Saaristo, A. et al., Oncogene 2000; 19:6122-6129), age-related macular degeneration (Otani, A. et al., Invest Opthalmol. Vis. Sci., 1999; 40:1912-1920), diabetic retinopathy (Watanabe, D. et al., Am. J. Opthalmol. 2005; 139:476-481), rheumatoid arthritis (Fearon, U. et al., J. Rheumatol. 2003; 30:260-268; Paleolog, E. M. et al., Arthritis Res. 2002; 4:S81-S90), psoriasis (Kuroda, K. et al., J. Invest Dermatol. 2001; 116:713-720), acute and chronic inflammation (McDonald, D. M. et al., Am. J. Respi. Cri. Care Med. 2001; 164:S39-S45; Roviezzo, F. et al., J. Pharmacol. Exp. Ther. 2005; 314: 738-744), atherosclerosis (Lim H S, et al., Atherosclerosis, 2005; 180:113-118) and lymphatic proliferative diseases such as tumor lymphangiogenesis (Scavelli, C. et al., Leukemia 2004; 18:1054-1058) and lymphatic metastasis (Sfiligoi, C. et al., Int. J. Cancer 2003; 103:466-474). Therefore, the present invention provides synchronous blockade of VEGF-A and Ang2, preferably with a decoy receptor, intradiabody (double antibody) or RNA interference for treating VEGF-A and/or Ang2-associated diseases.